Ethanol produced from lignocellulosics is an environmentally friendly alternative to fossil fuels. As a substantial fraction of lignocellulose material consists of xylose, it is necessary to ferment efficiently xylose to ethanol to make the process cost-effective [1].
Some yeasts, filamentous fungi and bacteria are able to convert xylose to ethanol. Yeasts and most of other fungi first reduce xylose to xylitol using xylose reductase, which strongly prefers NADPH as coenzyme, EC 1.1.1.21 (XR). Then they oxidize xylitol to xylulose with strictly NAD-dependent xylitol dehydrogenase, EC 1.1.1.9 (XDH) [2]. The difference in cofactor specificity results in redox imbalance that leads to decreasing ethanol production and accumulation of xylitol [3, 4, 5, 6, 7, 8]. The xylitol production has been reduced by metabolic engineering directed to optimize the expression levels of XR and XDH [9, 10, 11, 12], change the cofactor specificity of XR from NADPH to NADH [13, 13a], or modify the redox metabolism of the host cell [14, 15, 16]. The other used approach to bypass redox imbalance during xylose fermentation was based on expression of fungal or bacterial xylose isomerase, EC 5.3.1.5 (XI) which converts xylose directly to xylulose and does not require redox cofactors [17, 18]
The additional overexpression of xylulokinase, EC 2.7.1.17 (XK) (the third enzyme in the xylose metabolism) that converts xylulose to xylulose-5-phosphate, which enters the pentose phosphate pathway and then into the central metabolism, has been shown to enhance both aerobic and anaerobic xylose utilization in XR-XDH- as well as XI carrying strains [12, 19]. Overexpression of XK is necessary to overcome the naturally low expression level of this enzyme [3, 5]. The overexpression resulted in more efficient conversion of xylose to ethanol [5, 20].
The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45-48° C.) [21, 22, 23]. This property of H. polymorpha makes it a good candidate for use in an efficient process of simultaneous saccharification and fermentation (SSF). SSF combines enzymatic hydrolysis of lignocellulosic material with subsequent fermentation of released sugars in the same vessel. Major advantages of exploring the utility of H. polymorpha for ethanol production from cellulosic material using are this yeast has (i) well developed methods of molecular genetics and (ii) the availability of a whole genome sequence for a model strain CBS4732 [24; 25].